Promoters and compositions

Abstract

The present invention relates to activatable promoters, as well as expression systems, vectors, delivery systems, reaction vessels, aqueous objects and compositions comprising said activatable promoters. The invention also provides compositions comprising assemblies and networks of aqueous objects comprising said activatable promoters. The invention additionally provides methods of expressing a transcript and methods of expressing a polypeptide using the activatable promoters of the invention. The invention further provides phospholipid mixtures, as well as aqueous objects and compositions comprising said phospholipid mixtures.

The invention claimed is: 
     
       1. An activatable promoter comprising a nucleotide promoter sequence and a transcription inhibition moiety, wherein:
 a) the transcription inhibition moiety comprises a complex of a protein and small molecule; 
 b) the small molecule is coupled to a component of the nucleotide promoter sequence via a photocleavable moiety, wherein the component of the nucleotide promoter sequence is one or more nucleotide bases or nucleotide base analogues, and wherein the one or more nucleotide bases or nucleotide base analogues comprises a thymine; 
 c) the photocleavable moiety comprises a nitroaryl group and a diamine linker, and the transcription inhibition moiety and the photocleavable moiety are coupled to the thymine via the diamine linker, and the small molecule is coupled to the diamine linker via the nitroaryl group; 
 d) the diamine linker and thymine form a C6-amino-dT or a C6-amino-T; and 
 e) the nucleotide promotor sequence is configured to be activated when the transcription inhibition moiety is uncoupled from the activatable promoter by cleavage of the photocleavable moiety. 
 
     
     
       2. The activatable promoter according to  claim 1 , wherein the nitroaryl group of the photocleavable moiety is 2-nitrobenzyl. 
     
     
       3. The activatable promoter according to  claim 1 , wherein the small molecule is biotin and the protein is streptavidin. 
     
     
       4. An expression system comprising the activatable promoter according to  claim 1  operably linked to an open reading frame or a gene. 
     
     
       5. The expression system according to  claim 4 , wherein the open reading frame or gene encodes a membrane protein. 
     
     
       6. The expression system according to  claim 5 , wherein the membrane protein is an a-hemolysin (aHL) pore protein. 
     
     
       7. The expression system according to  claim 5 , wherein the membrane protein comprises a pump, a channel, a pore, a receptor protein, a transporter protein, or a protein which effects cell recognition or a cell-to-cell interaction. 
     
     
       8. The expression system according to  claim 4 , wherein the system is operably configured for use with bacteriophage transcription components. 
     
     
       9. The expression system according to  claim 4 , wherein the nucleotide promoter sequence comprises a T7 promoter. 
     
     
       10. The expression system according to  claim 4 , wherein the system is operably configured for use with prokaryotic transcription components. 
     
     
       11. The expression system according to  claim 10 , wherein the prokaryotic transcription components are bacterial transcription components. 
     
     
       12. The expression system according to  claim 4 , wherein the system is operably configured for use with eukaryotic transcription components. 
     
     
       13. The expression system according to  claim 12 , wherein the system is operably configured for use with insect or mammalian transcription components. 
     
     
       14. The expression system according to  claim 4 , further comprising one or more additional transcriptional elements. 
     
     
       15. The expression system according to  claim 14 , wherein the transcriptional element is an enhancer. 
     
     
       16. A vector comprising an expression system according to  claim 4 . 
     
     
       17. The vector according to  claim 16 , wherein the vector is a linear double-stranded DNA molecule or a continuous double-stranded DNA molecule. 
     
     
       18. The vector according to  claim 16 , wherein the vector is a viral vector. 
     
     
       19. A delivery system comprising a vector according to  claim 18 , wherein the delivery system comprises a viral delivery system. 
     
     
       20. The vector according to  claim 18 , wherein the vector is a retrovirus, lentivirus, adenovirus, adeno-associated virus or herpes simplex virus vector. 
     
     
       21. A delivery system comprising the vector according to  claim 16 , wherein the delivery system comprises a yeast system, a lipofection system, a microinjection system, a biolistic system, virosomes, liposomes, immunoliposomes, polycations, lipid: nucleic acid conjugates or artificial virions. 
     
     
       22. A method of expressing a transcript, the method comprising: contacting the activatable promoter according to  claim 1  with an uncoupling agent, the agent being capable of uncoupling the transcription inhibition moiety from the component of the nucleotide promoter sequence; whereupon the transcript is expressed following uncoupling. 
     
     
       23. The method according to  claim 22 , wherein the activatable promoter is comprised in an expression system or wherein the activatable promoter is comprised in a vector. 
     
     
       24. The method according to  claim 22 , wherein the uncoupling agent is electromagnetic radiation. 
     
     
       25. The method according to  claim 24 , wherein the nitroaryl group of the photocleavable moiety is 2-nitrobenzyl. 
     
     
       26. The method according to  claim 24 , wherein the electromagnetic radiation is ultra violet (UV). 
     
     
       27. The method according to  claim 24 , wherein the electromagnetic radiation is infra-red (IR). 
     
     
       28. The activatable promoter according to  claim 1 , wherein the small molecule is digoxigenin (DIG) and the protein is a DIG antibody. 
     
     
       29. The activatable promoter according to  claim 1 , wherein the nitroaryl group is a nitrobenzyl group.